ON HEMOCHROMOGEN

1. Every hemochromogen consists of the iron pyrrol complex, reduced heme, combined with some nitrogenous substance. 2. In every hemochromogen there is the equilibrium: Hemochromogen ⇄ Reduced heme + Nitrogenous substance. 3. Cyanide can form two distinct compounds with reduced heme, one of which is the typical hemochromogen, cyan-hemochromogen. 4. Reduced heme in alkaline solution has a great affinity for cyanide. 5. Cyan-hemochromogen probably contains one cyanide group per heme. 6. The hemochromogen prepared from hemoglobin is a compound of denatured globin and reduced heme. 7. The individual molecule of denatured globin, of hypothetical molecular weight 16,700, can convert at least 10 molecules of reduced heme into hemochromogen. 8. The hemochromogen-forming capacity of globin is, under given conditions, greater than that of edestin, which in turn, is greater than that of zein.     

ON PYRIDINE HEMOCHROMOGEN

1. Cyanide hemochromogen probably contains one cyanide group per heme group. 2. The equilibrium between pyridine hemochromogen and its components, pyridine and reduced heme, is complicated to an unknown extent by the precipitation of reduced heme and the aggregation of pyridine hemochromogen. 3. These complications were not taken into account in R. Hill''s experiments on pyridine hemochromogen. 4. Even if Hill''s experiments are sound they do not prove his conclusion that pyridine hemochromogen contains two pyridine groups per heme group.     

Structure of cyanide methemoglobin

X-ray difference Fourier analysis at 2.8 Å resolution shows that the tertiary structures of horse cyanide methemoglobin and methemoglobin differ significantly. The conformations of the heme groups and their interactions with the globin are altered. Short contacts with globin side chains affect cyanide binding to the hemes, and the changes in globin-ligand contact upon substitution of cyanide for water in turn directly affect globin structure. Although the ligand peaks lie off the heme axes, the atoms FeCN may still lie on a straight line (as they do in small iron cyanide complexes), with this line not normal to the mean heme plane. This linear binding configuration is consistent with the observed motion and deformation of the porphyrin. Although motion of the iron atoms is not directly apparent, there is evidence that some changes in tertiary structure are induced by shortening of the iron-pyrrol nitrogen bond lengths. This and other studies suggest that the structural changes responsible for co-operativity in hemoglobin may be initiated not merely by an alteration in the covalent porphyrin-proximal histidine linkage, but by changes in the noncovalent interactions of the globin with the ligand and porphyrin as well.     

Complexes of metal phthalocyanines with globin as the models of heme proteins.

The reaction between iron and cobalt tetrasulfonated phthalocyanines and globin results in the formation of the green complexes, as has been proved by difference spectroscopy. Spectrophotometric titration data indicate the formation of those complexes at the molar ratio 1:1. The complexes of ferrous, ferric and cobaltous tetrasulfonated phthalocyanines with globin have been isolated from the reaction mixtures by separation on Sephadex G-50 and precipitation of the protein fractions with ammonium sulfate. The visible spectra of these complexes are characterised by the main intensive peak at 641 nm, 678 nm, and 675 nm for ferric, ferrous and cobaltous derivatives, respectively. The new globin complexes have the property of reversible combination with oxygen and coordination with cyanide ions. It is evidence from the results of the spectrophotometric titrations of hemoglobin and methemoglobin with cobaltous tetrasulfonated phthalocyanine that iron protoporphyrins are displaced by this cobalt derivative; this suggests that phthalocyanine and porphyrin are bonded in a similar manner.     

Ferrous Campylobacter jejuni truncated hemoglobin P displays an extremely high reactivity for cyanide - a comparative study

Campylobacter jejuni hosts two hemoglobins (Hbs). The Camplylobacter jejuni single-domain Hb (called Cgb) is homologous to the globin domain of flavohemoglobin, and it has been proposed to protect the bacterium against nitrosative stress. The second Hb is called Ctb (hereafter Cj-trHbP), belongs to truncated Hb group III, and has been hypothesized to be involved in O(2) chemistry. Here, the kinetics and thermodynamics of cyanide binding to ferric and ferrous Cj-trHbP [Cj-trHbP(III) and Cj-trHbP(II), respectively] are reported and analyzed in parallel with those of related heme proteins, with particular reference to those from Mycobacterium tuberculosis. The affinity of cyanide for Cj-trHbP(II) is higher than that reported for any known (in)vertebrate globin by more than three orders of magnitude (K = 1.2 x 10(-6) m). This can be fully attributed to the highest (ever observed for a ferrous Hb) cyanide-binding association rate constant (k(on) = 3.3 x 10(3) m(-1).s(-1)), even though the binding process displays a rate-limiting step (k(max) = 9.1 s(-1)). Cj-trHbP(III) shows a very high affinity for cyanide (L = 5.8 x 10(-9) m); however, cyanide association kinetics are independent of cyanide concentration, displaying a rate-limiting step (l(max) = 2.0 x 10(-3) s(-1)). Values of the first-order rate constant for cyanide dissociation from Cj-trHbP(II)-cyanide and Cj-trHbP(III)-cyanide (k(off) =5.0 x 10(-3) s(-1) and l(off) > or = 1 x 10(-4) s(-1), respectively) are similar to those reported for (in)vertebrate globins. The very high affinity of cyanide for Cj-trHbP(II), reminiscent of that of horseradish peroxidase(II), suggests that this globin may participate in cyanide detoxification.     

Purification and characterization of catalase-1 from Bacillus subtilis

The catalase activity produced in vegetative Bacillus subtilis, catalase-1, has been purified to homogeneity. The apparent native molecular weight was determined to be 395,000. Only one subunit type with a molecular weight of 65,000 was present, suggesting a hexamer structure for the enzyme. In other respects, catalase-1 was a typical catalase. Protoheme IX was identified as the heme component on the basis of the spectra of the enzyme and of the isolated hemochromogen. The ratio of protoheme/subunit was 1. The enzyme remained active over a broad pH range of 5-11 and was only slowly inactivated at 65 degrees C. It was inhibited by cyanide, azide, and various sulfhydryl compounds. The apparent Km for hydrogen peroxide was 40.1 mM. The amino acid composition was typical of other catalases in having relatively low amounts of tryptophan and cysteine.     

Respiratory cytochromes of Euglena gracilis

1. 1. Difference spectra of whole cells and of a particulate fraction of a streptomycin-bleached strain of Euglena gracilis showed the presence of a b-type cytochrome, cytochrome b (561 Euglena), and an a-type cytochrome, cytochrome a-type (609 Euglena). The cytochromes were characterized by pyridine hemochromogen formation and were found associated with a particulate fraction enriched with mitochondria.

2. 2. Both b-type and a-type cytochromes were reduced by succinate, oxidized by oxygen and reacted with a soluble c-type cytochrome, cytochrome c-type (556 Euglena), in reversible oxidation-reduction reactions. The steady-state level of reduction for each cytochrome was 92, 22 and 5% of the anaerobic level for the b-type, c-type and a-type cytochrome, respectively.

3. 3. Oxidation of c-type and a-type cytochromes was completely inhibited by cyanide, although respiration of a particulate fraction was only 60% inhibited by the same concentration of cyanide. Antimycin A inhibited respiration by up to 70% but completely inhibited reduction of the c-type cytochrome.

4. 4. The data suggest that electron transfer in the respiratory pathway of Euglena involves the b-, c- and a-type cytochrome in a direct sequence. The cyanide and antimycin A-insensitive oxidation pathway is considered to involve a more direct oxidation of the b-type cytochrome.

Optical and magnetic resonance studies of formate binding to horse liver catalase and sperm whale myoglobin.

The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.     

Cloning and amplification of alpha and beta mouse globin gene sequences synthesised in vitro.

New chimeric Escherichia coli plasmids containing alpha or beta globin gene sequences of the mouse were constructed. Double-stranded DNA, synthesised in vitro in a 2-step reaction from mouse globin mRNA was inserted into E. coli plasmid pCR1, after tailing of the 2 DNAs with dG and dC respectively. Some of the mouse globin plasmids described contain at least 90% of the globin mRNA sequence and therefore contain the entire translated sequence of the globin genes. Some possible uses of these recombinant plasmids are described.     

Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical

Previous studies established that the cyanyl radical ((*)CN), detected as 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(*)CN by the electron spin resonance (ESR) spin-trapping technique, can be generated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H(2)O(2)) and by mitochondrial cytochrome c oxidase (CcO) in the absence of H(2)O(2). To investigate the mechanism of inhibition by cyanyl radical, we isolated and characterized the iron protoporphyrin IX and heme a from the reactions of CN(-) with HRP and CcO, respectively. The purified heme from the reaction mixture of HRP/H(2)O(2)/KCN was unambiguously identified as cyanoheme by the observation of the protonated molecule, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser desorption/ionization (MALDI) mass spectrum. The proton NMR spectrum of the bipyridyl ferrous cyanoheme complex revealed that one of the four meso protons was missing and had been replaced with a cyanyl group, indicating that the single, heme-derived product was meso-cyanoheme. The holoenzyme of HRP from the reconstitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no detectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoHRP was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, the heme a isolated from partially or fully inhibited CcO did not show any change in the structure of the protoporphyrin IX as indicated by its MALDI mass spectrum, which showed an (M + H)(+) of m/z = 853.6, and by its pyridine hemochromogen spectrum. However, a protein-centered radical on the CcO can be detected in the reaction of CcO with cyanide and was identified as the thiyl radical(s) based on inhibition of its formation by N-ethylmaleimide pretreatment, suggesting that the protein matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In addition to the difference in heme structures between HRP and CcO, the available crystallographic data also suggested that the distinct heme environments may contribute to the different inhibition mechanisms of HRP and CcO by cyanyl radical.     

Role of bacterial cyanide production in differential reaction of plant cultivars to deleterious rhizosphere pseudomonads

Lettuce and wheat cultivars, differing in reaction to root inoculation with plant growth-inhibitory bacteria, were tested for sensitivity to (i) gaseous metabolites produced by deleterious, cyanogenic isolates of Pseudomonas fluorescens, and to (ii) pure cyanide. Reactions were read as shoot and/or root elongation after exposure of seedlings to the volatiles in vitro. Lettuce cultivar Salad Bowl was significantly less sensitive than cv. Montana, both to bacterial volatiles and to pure cyanide, and a similar difference between these cultivars was also obtained in greenhouse experiments where bacteria were inoculated directly on the roots. Cultivar differences were, however, not recorded, either in vitro or in the greenhouse, when the bacteria were grown on a medium which did not support cyanide production. In wheat, a difference in sensitivity to bacteria-produced volatiles was recorded between two cultivars (Drabant and Besso) differing in reaction to bacterial inoculation, but in contrast to lettuce cultivars, the wheat cultivars tested did not react differentially to pure cyanide. The results suggest that in lettuce differential sensitivity to cyanide is one factor behind cultivar differences in reaction to the bacteria tested, even though bacterial metabolites other than cyanide may contribute to the plant growth inhibition recorded. In wheat, however, differential cultivar responses to these bacteria could not be shown to be related to cyanide.     

Synergistic effect of NADH on NADPH-dependent acetaminophen activation in liver microsomes and its inhibition by cyanide

The effects of NADH and cyanide on NADPH-dependent acetaminophen activation in rat and mouse liver microsomes were studied. In both rat and mouse microsomes, NADPH-dependent acetaminophen-glutathione conjugate production was synergistically enhanced by the addition of NADH, whereas NADH alone did not initiate this reaction. The data suggest that the second electron in this reaction may be transferred from NADH. The present findings are different from a previous report in a reconstituted system that NADH decreases covalent binding of acetaminophen to proteins. This reaction was inhibited by low concentrations of sodium cyanide. The role of the cyanide sensitive factor in this reaction in liver microsomes remains to be further clarified.     

Inhibition of heme biosynthesis in erythroid precursors (BFU-e) isolated from human peripheral blood: effects on globin synthesis

We studied the relationship between heme accumulation and globin synthesis in human erythroid precursors which were stimulated by 2 I.U. of erythropoietin in semi-solid cultures (1% methyl-cellulose, 20% fetal calf serum) and treated with 6-9 micrograms/ml of desferrioxamina (DF), a potent inhibitor of heme synthesis (6). Heme accumulation was detected by specific reaction with benzidine (4), globin synthesis by CM-cellulose column chromatography. Our results demonstrate that globin gene expression occurs in DF-treated erythroid cells which do not accumulate heme molecules. As heme does affect translation and stability of globin mRNA (10) our system might be suitable for studies focused on pathological alterations of erythropoiesis associated with the presence of unstable globin mRNAs and/or unstable globins.     

Precipitation of gold(I) cyanide from dicyanoaurate solutions by Acinetobacter RFB1

Summary The precipitation of gold(I) cyanide by removal of one cyanide group from dicyanoaurate by Acinetobacter RFB1 is reported. In the absence of essential salts but in the presence of ferrous ions, precipitation of gold(I) cyanide could be achieved without growth of the microorganism. Maximal precipitation was also dependent on the level of dissolved oxygen in the reaction media. Silver cyanide and other metal cyanide salts could not be precipitated as effectively as gold(I) cyanide. This system may find application in the separation of gold from other metal cyanide complexes in mining eluants.Offprint requests to: I. Finnegan     

A combined micro-resonance Raman and absorption set-up enabling in vivo studies under varying physiological conditions: The nerve globin in the nerve cord of Aphrodite aculeata

We hereby report on the design of a set-up combining micro-resonance Raman and absorption spectroscopy with a microfluidic system. The set-up enabled us to study the nerve globin of Aphrodite aculeata in the functional isolated nerve cord under varying physiological conditions for extended periods of time. The oxygenation cycle of the organism was triggered by utilizing the microfluidic system that allowed for a fast switch between aerobic and anaerobic conditions. The nerve globin was found to very easily shift from a penta-coordinated high spin ferrous form to the oxy state upon a change from anaerobic to aerobic conditions. The observed fast reaction to varying O(2) concentrations supports an oxygen-carrying and/or -storing function of the nerve globin. In addition, by combining resonance Raman and absorption spectroscopy, the physiological response could be distinguished from light-induced effects.     

Histochemical observations on endogenous lactoperoxidase activity of the bovine submandibular salivary gland

Synopsis Seromucous demilunar cells of glutaraldehyde-fixed bovine submandibular salivary glands are intensely stained when sections are incubated in a benzidine-or a 3,3-diaminobenzidine-hydrogen peroxide medium in the pH range 6.0–9.0 whereas mucous acinar cells are completely unreactive. The histochemical reaction is completely inhibited by 3-amino-1,2,4-triazole. In contrast 2,4-dichlorophenol or potassium cyanide has little or no effect on the staining of demilunar cells. Striated duct cells also display a positive reaction with the diaminobenzidine method; this staining reaction, however, is most intense at pH 6.0. Furthermore, this reaction is markedly affected by potassium cyanide. The positive histochemical benzidine and diaminobenzidine reactions of demilunar cells probably corresponds to endogenous lactoperoxidase activity. On the other hand, the positive reaction shown by striated ducts, with optimal staining at pH 6.0 and which is completely inhibited by potassium cyanide, seems to be due to cytochromal oxidation of diaminobenzidine.     

A direct evidence for the involvement of poly(A) in protein synthesis

A radioactive polyadenylated globin mRNA was translated in either rabbit reticulocyte lysate or wheat germ extract under various conditions. When globin mRNA was translated, globin synthesis was directly proportional to the rate of loss in A units from the poly(A) tail. On the other hand, when globin poly(A) mRNA was incubated under non-translated conditions, no loss of A units was detected. The presence of ribonuclease inhibitor in the reaction mixture did not alter either the rate of globin synthesis or the loss in A units from the poly(A) tail. The present data suggests a correlation between protein synthesis and loss in A units from the poly(A) tail.     

Modification by cyanide of the aniline-binding reaction with cytochrome P-450.

Hydroxylation of aniline to p-aminophenol catalyzed by the cytochrome P-450-containing monooxygenase system of liver microsomes is inhibited by cyanide, but microsomal NADPH-cytochrome c reductase is insensitive to this inhibitor. The interaction of aniline with membrane-bound cytochrome P-450, according to spectrophotometric analyses, consists of two phases with respect to aniline concentration, and cyanide interferes differently with these two reaction phases. Noncompetitive and competitive (or mixed type) inhibitions of the aniline-binding reaction by cyanide are observed in reaction systems containing low and, high concentrations of aniline, respectively, a situation similar to the inhibitory action of cyanide on aniline hydroxylase activity. Abnormal aniline-induced difference spectra appeared when cyanide was added as the spectral modifier, and the magnitude of the spectral change in the presence of both aniline and cyanide was a nonadditive change. These results suggest the dissociation of the cytochrome P-450·cyanide complex by aniline. A similar result indicating dissociation of the complex was also obtained by epr spectroscopy. We therefore suggest that addition of a high concentration of substrate causes insensitivity of the microsomal hydroxylase system to cyanide.     

Cyanide and sulfide interact with nitrogenous compounds to influence the relaxation of various smooth muscles

Sodium nitroprusside relaxed guinea pig ileum after the segment had been submaximally contracted by either histamine or acetylcholine, intact isolated rabbit gall bladder after submaximal contraction by either acetylcholine or cholecystokinin octapeptide, and rat pulmonary artery helical strips after submaximal contraction with norepinephrine. In each of these cases the relaxation produced by nitroprusside was at least partially reversed by the subsequent addition of excess sodium cyanide. Cyanide, however, in nontoxic concentrations did not reverse the spasmolytic effects of hydroxylamine hydrochloride, sodium azide, nitroglycerin, sodium nitrite, or nitric oxide hemoglobin on guinea pig ileum, nor did cyanide alone in the same concentrations have any effect. The similar interaction between nitroprusside and cyanide on rabbit aortic strips is not dependent on the presence of an intact endothelial cell layer. Also, on rabbit aortic strips and like cyanide, sodium sulfide reversed the spasmolytic effects of azide and hydroxylamine, but it had little or no effect on the relaxation induced by papaverine. Unlike cyanide, however, sulfide augmented the relaxation induced by nitroprusside, and it reversed the effects of nitric oxide hemoglobin, nitroglycerin, and nitrite. A direct chemical reaction between sulfide and nitroprusside may account for the difference between it and cyanide. Although evidence was obtained also for a direct chemical reaction between sulfide and norepinephrine, that reaction does not seem to have played a role in these results. These observations suggest the existence of at least three distinct subclasses of so-called nitric oxide vasodilators. At least in some cases cyanide and sulfide cannot be acting by the same mechanism in their modifications of the responses to the agonists.